Viral vector coding for juvenile hormone esterase

ABSTRACT

A diagnostic or control composition is useful to characterize or control insects and comprises a nucleotide sequence coding for juvenile hormone esterase (JHE). The coding sequence may be combined with a promoter sequence regulating the transcription thereof in a recombinant expression vector for use in controlling insects having a juvenile hormone esterase dependency. Preferred embodiments of the invention are recombinant baculoviruses in which a mutated JHE coding sequence provides relatively rapid speed of kill in insects.

This invention was made with Government support under NIH Grant No.ES-02710, NSF Grant No. DCB-85-18697 and USDA Grant No. 85-CRCR-1-1715.

This is a division of application Ser. No. 07/927,851, filed Aug. 10,1992 U.S. Pat. No. 5,643,776, which is a continuation-in part of U.S.Ser. No. 07/725,226, filed Jun. 26, 1991, now abandoned, which was acontinuation of Ser. No. 07,265,507, filed Nov. 1, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention relates to uses of nucleotide sequences coding forjuvenile hormone esterase, and more particularly to recombinantexpression vectors including juvenile hormone esterase or mutant codingsequences having uses such as in controlling insects.

BACKGROUND OF THE INVENTION

The lepidopteran family noctuidae includes some of the most destructiveagricultural pests, such as the genera Heliothis, Helicoverpa,Spodoptera and Trichoplusia. For example, included in this family arethe tobacco budworm (Heliothis virescens), the cotton leafworm (Alabamaargillacea), the spotted cutworm (Amathes c-nigrum), the glassy cutworm(Crymodes devastator), the bronzed cutworm (Nephelodes emmedonia), thefall armyworm (Laphygma frugiperda), the beet armyworm (Spodopteraexigua) and the variegated cutworm (Peridroma saucia). Juvenile hormoneesterase is responsible for the stage-specific metabolism of juvenilehormone in such insects.

Juvenile hormone and juvenile hormone esterase have been studiedextensively in the Lepidoptera. In the final larval growing stage ofthese insects, there is a rapid decline in the juvenile hormone titerwhich initiates the physiological and behavioral events precedingpupation and adult development. This decline in the juvenile hormonetiter appears to be regulated by an increase in degradation by juvenilehormone esterase as well as a reduction of biosynthesis. Juvenilehormone esterase activity is very low in the early stadia of larvalgrowth. Even at the peak activity levels in the blood of the finalstadium, the concentration of juvenile hormone esterase has beenestimated at less than 0.1 percent of the total protein. Yet the enzymehas a high affinity for juvenile hormone.

The initial reduction in juvenile hormone titer in the last larvalstadium initiates a sequence of events leading to pupation. Powerful andselective chemical inhibitors of juvenile hormone esterase have beenused in vivo to demonstrate the developmental consequences of blockingthe activity of juvenile hormone esterase. For example, a group of thechemical inhibitors of juvenile hormone esterase are thetrifluoromethylketone sulfides, as described by U.S. Pat. No. 4,562,292,issued Dec. 31, 1985, inventors Hammock et al. Treatment in the finallarval stadium of the tomato hornworm (Manduca sexta) and other mothlarvae with potent inhibitors can block almost all of the blood juvenilehormone esterase activity and cause a delay in the time ofmetamorphosis, presumably by allowing juvenile hormone to remainpresent.

Classical methods of protein purification have been inefficient for thelarge-scale purification of juvenile hormone esterase because theesterase is in picomole amounts even at its peak levels. More recently,a purification method for juvenile hormone esterase from larval bloodhas been developed. Abdel-Aal and Hammock, Science, 233, pp. 1073-1076(1986).

There is a need for genes which when produced in expression systems willlead to rapid insect death, disruption of development, and/or cessationof feeding. Recent efforts have centered on the insect specific toxinsfrom Bacillus thuringiensis (Merryweather et al., 1990), from thescorpions Buthus eupeus (Carbonell et al., 1988) and Androctonusaustralis (Stewart et al., 1991; McCutchen et al., 1991; Maeda et al.,1991) and from the mite Pyemotes tritici (Tomalski and Miller, 1991).

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a recombinantexpression vector that halts feeding and disrupts the development ofinsect pests by artificial expression of juvenile hormone esterase at anearly stage of development.

One aspect of the present invention comprises a vector with a codingsequence capable of expressing a protein in a host cell expressionsystem. When expressed, the protein degrades, binds with or sequestersjuvenile hormone or a juvenile hormone homolog within the host.

A preferred use of the inventive vectors is for expression in insects byinfecting susceptible host insect cells with a recombinant baculovirusso that insect development is disrupted. Improved expression of JHE in abaculovirus system (with an improved lethal dose value) has beenprovided by mutagenesis. This improved expression is important becauseexcessive insect feeding (before the virus acts on the insect pest) canresult in unacceptable crop damage. Our improved vectors achieve earlierinsect kill.

In another aspect of the present invention a transgenic, non-humanorganism has a nucleotide sequence introduced therein. The nucleotidesequence preferably is substantially similar to the cDNA illustrated bySEQ ID NOS:1 or 2 or to the gene illustrated by FIG. 6. These sequencesare effective to alter at least one determinable property of theorganism when expressed. A preferred altered property is enhanceddegradation of a methyl ester containing compound, such as disruption ofjuvenile hormone expression.

In another aspect of the present invention, the recombinant expressionvectors capable of expression in a host insect or plant cell includecoding sequences for a mutant or chimeric protein of a juvenile hormoneesterase and a promoter that is heterologous with the coding sequence.

These and other aspects and advantages of the present invention will nowbe more fully described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a restriction map for a cDNA coding for juvenilehormone esterase;

FIG. 2 illustrates construction of a plasmid embodiment in accordancewith the invention (designated pAcUW21.JHE);

FIG. 3 graphically illustrates the expression of wild type and threemodified JHEs, which are inventive embodiments, in baculovirus infectedSf cells in vitro;

FIG. 4 graphically illustrates the expression of JHE in the system usedfor the FIG. 3 data (baculovirus infected Sf cells in vitro), but withthree different promoters as additional inventive embodiments;

FIG. 5 graphically illustrates the in vivo expression of wild type andseveral modified JHEs post-injection of recombinant baculoviruses intosecond instar Manduca sexta; and

FIG. 6 summarizes the JHE gene itself.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The lepidopteran family Noctuidae includes a number of economicallydestructive agricultural pests. Briefly, two epithelial hormones controlmetamorphosis in such insects. Asteroid hormone, 20-hydroxyecdysone,causes the molt, while a terpenoid hormone, juvenile hormone (JH),determines the nature of the molt. If juvenile hormone titers are high,then the molt is isometric to a larger larval stage. If juvenile hormonetiters are low, then anisometric molt to a pupal stage will occur. Thus,the initial reduction in juvenile hormone titer in the last larvalstadium is a key event in insect development in that it initiates asequence of events leading to pupation and early in this sequence ofevents is the cessation of feeding. This reduction in juvenile hormonetiter is accomplished by a reduction of biosynthesis and a tremendousincrease in the highly aggressive enzyme known as juvenile hormoneesterase (JHE).

Juvenile hormone esterase (JHE) is biochemically interesting in that itis an extraordinarily efficient enzyme. The very low K_(m) 6×10⁻⁸ M ofjuvenile hormone esterase, its relatively high k_(CAT), and the lowmolarity of the enzyme even in the last larval stadium indicate thatproduction of even small amounts of juvenile hormone esterase willoverpower the ability of the corpora allata to make juvenile hormone(JH). The destruction of the juvenile hormone will be accelerated by thejuvenile hormone hemolymph carrier protein. This carrier protectsjuvenile hormone at earlier stages of development, but it binds juvenilehormone less tightly than the esterase (K_(d) =6.1×10⁻⁷ M for H.virescens), and thus serves to accelerate degradation of juvenilehormone by juvenile hormone esterase and keeps juvenile hormone out oflipophilic depots.

Under V_(max) conditions, the juvenile hormone esterase present in asingle fifth stadium noctuid larva could hydrolyze over 100,000 times asmuch juvenile hormone each minute as is present in an entire larva atany time during development. Under physiological conditions, juvenilehormone esterase acts as an infinitely large sink capable of extractingjuvenile hormone and other susceptible substrates from lipid depots andcarriers by mass action and instantly inactivating it. Thus, precociousappearance of juvenile hormone esterase will reduce juvenile hormonetiters, typically resulting in irreversible termination of the feedingstage, attempted pupation and death of the pest insect.

Juvenile hormone esterase is an insect protein which appears at criticaltimes in the insect's life. It appears to present no risk to othergroups of organisms. It is nonlethal to an individual cell which allowsand perhaps encourages viral replication; yet the enzyme will fatallydisrupt the normal development of the organism. Because the substrate(juvenile hormone) readily penetrates membranes, the juvenile hormoneesterase need only be expressed in a few cells to deplete juvenilehormone. Differential tissue depletion of juvenile hormone is likely tobe even more rapidly fatal to an insect than uniform depletion.

Numerous attempts have been made to purify juvenile hormone seterases,but purification of the low abundance enzyme from a small tissue sourcehas proven very tedious. U.S. Pat. No. 5,098,706, issued Mar. 24, 1992,and filed concurrently with Ser. No. 07/265,507 of which this is acontinuation-in-part, incorporated by reference, exemplifies theadministration of an affinity purified enzyme to insects which resultsin anti-juvenile hormone activity. Such anti-juvenile hormone activityis effectively lethal, for example in blocking damage by herbivorousinsects.

One aspect of the present invention concerns uses of the coding sequencefor juvenile hormone esterase. SEQ ID NO:1 sets out the coding sequencefor one cDNA of JHE, and SEQ ID NO:2 sets out the coding sequence foranother. These coding sequences are for juvenile hormone esterase fromHeliothis virescens, although there is homology to Helicoverpa zea(formerly Heliothis zea), to Tricoplusia ni and (at lower stringency)hybridization to Manduca sexta. Further, JHE isolated (or derived) fromHeliothis (Helicoverpa) viresens functions to hydrolyze every known formof JH. This means that a coding sequence for JHE derived from H.virescens can be used to isolate the gene or the message from a varietyof species. FIG. 6 illustrates the JHE gene for H. virescens and gives,among other things, restriction enzyme sites.

The pattern of JHE activity and low message abundance suggests thatexpression of the enzyme is tightly regulated, perhaps at the level oftranscription. In Lepidoptera, the structure and transcriptional controlof developmentally regulated genes has been investigated, but most ofthese genes are multiple copy and the mRNA is abundant. By contrast, JHEfrom H. virescens apparently is encoded by the single gene illustratedby FIG. 6, which produces a low-abundance message.

JHE Coding Sequence Cloning

As will also be described in Examples 1 and 2, the clone containing thesequence of the mRNA transcript of juvenile hormone esterase fromHeliothis virescens was isolated from a lambda gt-11 expression library.To make the expression library, total RNA was isolated by homogenizingfat bodies in guanidinium thiocyanate and centrifugation through cesiumchloride. The fat bodies were from last instar larvae that had beentreated with epofenonane 24 hours previously. Poly-adenylated RNA wasprepared by one cycle of oligo-dT chromatography from which cDNA wassynthesized and size selected for greater than 1350 base pairs. The sizeselected cDNA was suitably processed and ligated to arms of alambda-gt11 phage expression vector. The ligated DNA was then packagedinto phage heads, infected into host cells and plated on a lawn of hostE. coli. The cDNA library was not amplified prior to screening.

Screening was done immunochemically on nitrocellulose filters to whichproteins from plated phage had been bound after induction of proteinsynthesis. Clones reacting with antibodies specific for juvenile hormoneesterase were plaque-purified after detection with immunohistochemicalmeans. A second round of screening was then conducted upon the isolatedclones with hybridization to a mixture of synthetic nucleotidescomplementary to the deduced mRNA sequence possibilities determined fromthe N-terminal amino-acid sequence of juvenile hormone esterase.

The amino acid sequence was determined by automated Edman degradation ofthe purified protein. From this round of screening, three 3,000 basepairclones, 3hv1, 3hv16 and 3hv21 were isolated, subcloned into plasmids andsubjected to restriction analysis. The later two clones have theirsequences given by SEQ ID NOS. 2 and 1. Their length matched the 3,000basepair length of the juvenile hormone esterase mRNA transcript asdetermined by Northern blotting with radiolabeled DNA of clone 3hv21. An840 basepair fragment of the 5' coding region of 3hv21 was thensequenced. The sequence confirmed the clone to be that coding forjuvenile hormone esterase as the deduced amino-acid sequence matched 33of the 35 of the doubly confirmed amino-acids sequenced at theN-terminus of the purified protein.

Uses of JHE Coding Sequence

The JHE coding sequence, various mutations and analogs and restrictionfragments thereof have a variety of uses. For example, the JHE codingsequence is useful in characterizing insects related to or within theHeliothis group. Thus, Example 3 describes the utility of the cDNA as adiagnostic tool. Once the insect has been characterized, in laboratoryor field, then appropriate steps for control may be taken.

In addition, the reduction in JH titer in some insects is accomplishedby a combination of reduction in biosynthesis and an increase indegradation by JHE. Thus, the JHE coding sequence can be used incombination with an agent that reduces the JHE biosynthesis orinfluences its distribution and kinetics.

Since the juvenile hormone esterase coding sequence (illustrated by SEQID NO:1 and SEQ ID NO:2) has been determined, methods to elicit theprecocious expression of juvenile hormone esterase for insect controlare now possible.

The recent development of a vital expression vector from a virus endemicin and selective for noctuid species permits practical field controlsystems for the Boll Worm and other insect pests. U.S. Pat. No.4,745,051, issued May 17, 1988, inventors, Smith, et al., incorporatedherein by reference describes a method for producing a recombinantbaculovirus expression vector capable of expression of a selected genein a host insect cell. The method exemplified by U.S. Pat. No. 4,745,051was used to express β-Interferon by infecting susceptible host insectcells with a recombinant baculovirus expression vector. Briefly,baculovirus DNA is cleaved to obtain a DNA fragment containing at leasta promoter of the baculovirus gene. One baculovirus gene is that codingfor polyhedrin, since the polyhedrin protein is one of the most highlyexpressed eucaryotid genes known, although other promoter and hybridpromoter sequences may be used.

Although the polyhedrin promoter can be used in preparing inventiveexpression vectors, we have found JHE can be produced even earlier andat still greater levels under the p10 and basic protein promoters, asillustrated by the data of FIG. 4. The preferred baculovirus utilized isAutographa californica , although other baculovirus strains may besuitably utilized. Autographa californica (AcNPV) is of particularinterest as various major pest species within the genera Spodoptera,Trichoplusia, and Heliothis are susceptible to this virus.

In the present invention, a baculovirus may be modified by the methoddescribed by U.S. Pat. No. 4,745,051, but by utilizing the juvenilehormone esterase coding sequence (or a mutant of JHE coding sequence)rather than the β-Interferon gene. A recombinant expression vector thuscomprises a JHE coding sequence or a JHE mutant under the control of apromoter sequence (such as the polyhedrin, p10 or basic proteinpromoters), which is heterologous with the JHE coding sequence and whichregulates the transcription thereof. Expression of the JHE codingsequence gene is accomplished by infecting susceptible host insect orplant cells so as to produce precociously appearing juvenile hormoneesterase. In insect cells the JHE appearance, even at low levels,disrupts insect development. In plant cells the JHE appearance canprovide protection against insect predators.

Aspects of the present invention will be illustrated by a number ofexamples. Examples 1-3 primarily describe work obtaining cDNA for JHEand some characterizations for the JHE gene. Example 4 describespreparation of improved baculovirus (particularly useful as insecticidalagents) by forming mutants of the original JHE nucleotide sequences, andalso discusses various other mutations that can be made. Example 5describes preparation of inventive vector embodiments with severaldifferent promoters. Example 6 describes further characterizations ofthe JHE gene itself.

Preparation of mutants

The degradation of proteins within cells occurs by many pathways, butthe best known are the ubiquitin and lysosomal pathways. For degradationby ubiquitin and recognition by many lysosomal enzymes, a protein musteither have free lysine (K) residues available on the surface of theprotein or appropriate lysine containing sequences respectively. Forubiquitin degradation, appropriate lysines are often found near theN-terminus of the protein. JHE contains a lysine at position 29 and hastryptophan as the N-terminal amino acid, which is a possible susceptibleresidue for targeting a protein to ubiquitin degradation. In addition,lysine residues occurring in a region rich in proline, glutamate,serine, and threonine are important for both ubiquitin and lysosomalprotease recognition. JHE has one such lysine at position 522. Theselysines were changed to arginine (R) (K29R and K522R) by site-directedmutagenesis, and then the individual mutants and the combined mutant(K29R+K522R) were tested in the baculovirus system for insecticidalactivity.

In addition, an inactive JHE may bind and sequester juvenile hormonerather than degrade it and thus be more effective than active JHE. Thus,natural insect proteins when modified by these methods can havedeleterious effects on the insect. A catalytic site mutation of serine201 to glycine was made (S201G) and tested for insecticidal activity.

The double lysine mutant (sometimes referred to as "KK" and sometimes"29,522" and sometimes as "K29R, K522R") and the catalytic serine mutant(S201G) of recombinant JHE enhance the ability of the AcNPV to kill inboth Heliothis virescens and Trichoplusia ni. The insecticidal activityof the catalytically deficient S201G mutant of JHE is unusual andpossibly contradictory to the current theories of JHE action. The timesrequired for 50% death of test insects are similar to those required forscorpion toxins that have recently been reported in the literature,engineered in A.cal NPV, and are a considerably safer alternative. Theseviruses can be produced easily in both tissue culture and in laboratoryor industrial insect colonies, unlike their toxin containingcompetitors, and represent a minor variation of a protein that occursnaturally in the insects. As this enzyme occurs in all insects, theengineering of this JHE into NPVs with differing host specificity allowsa wide spectrum of pests to be controlled in a rapid and effectivemanner.

In addition to the particular in vitro site-directed mutagenesistechnique described by Example 4 to exemplify the mutation aspect of theinvention, there are numerous techniques for mutagenesis which may beemployed to improve utility. For instance, it is possible to employother nucleotide-mediated mutagenesis protocols and similar techniquescan be used to create deletions or insertions. Deletions or insertionsmay be systematically incorporated, either by linker-insertion,linker-scan, or nested deletion. For example, it is possible to randomlyinsert small linkers into the JHE sequence. Other techniques for lessstructured mutagenesis include the use of degenerate nucleotide pools,misincorporation by DNA polymerases and chemical mutagenesis. Standardrestriction and ligation techniques can be used to create largedeletions or chimeric enzymes. New PCR methods for mutagenesis couldalso be used to generate classes of mutations.

Whether by site-directed mutagenesis techniques or other mutagenesisprotocols, we consider sequences with significant homology to SEQ IDNO:1 or SEQ ID NO:2 or to the JHE gene itself (illustrated by FIG. 6) tobe within the scope of this invention. By "significant similarity orhomology" is meant that the nucleotides have greater sequence similaritythan by chance alone and that the protein coded by the sequence, whenexpressed in an appropriate host cell expression system, degrades, bindswith, or sequesters juvenile hormone or those endogenous proteinscommonly recognized as juvenile hormone homologs due to their structuralsimilarity to JH.

Another way of looking at coding sequences that are substantially thesame as a juvenile hormone esterase coding sequence are those where thephysiological or biological action is as has just been described and theprotein expressed by the coding sequence reacts with a methyl estercontaining compound. For example, the JHE of H. virescens and that ofseveral other species all have high specificity for JH with a highk_(CAT) /K_(M) ratio (>10⁸). Juvenile hormone itself is, of course, amethyl ester containing compound (and includes an epoxidefunctionality), and of interest her enzymes of interest where expressionof the coding sequence leads to enhanced degradation of JH. For example,epoxide hydrolases are known to act similarly to JHE by hydrolysis of JHto yield biologically inactive diols.

Example 4 illustrates use of mutagenesis in baculovirus expressing JHEas an insecticidal agent, which are improved over recombinantbaculoviruses expressing JHE, since several mutants have resulted inimproved properties (e.g. reducing the lethal dose significantly inHeliothis virescens). For example, a double mutant has resulted inlethal dose improvements so that insects are killed about one day or oneand one half days faster than kill rates with the wild type virus. Thisis significant for crop protection because the plant damage done tocrops is a function of time that the crops are vulnerable to theinsects.

Example 5 illustrates JHE activity, in vitro under the control ofseveral different viral promoters. The expression level of JHE with abasic protein promoter is particularly exciting because greatlyincreased JHE expression (at levels up to about 5 times higher) havebeen demonstrated. This is particularly surprising because use of thebasic protein promoter with other coding sequences has indicated loweredexpression rather than enhanced expression of the protein coded by thecoding sequence. We believe the striking difference in expression levelslies in the fact that JHE is a packaged, or secreted, protein. As aconsequence, increased expression levels of secreted or exportedproteins should be achievable by combining the basic protein promoterwith a nucleotide coding sequence coding for the secreted or exportedprotein. The sequence and composition of the 19 residues prior to theNH₂ terminal Trp of the secreted major form of JHE match well with theconsensus for other signal peptides for secretion.

Modified, or mutated, JHEs in accordance with the invention are notlimited to just the H. virescens insect because the JHE enzyme iscapable of hydrolyzing all known JH homologs with similar k_(CAT) /K_(m)ratios, as well as related methyl esters. This means that JHE and JHEmutants are applicable in many expression systems, and it is clear thatJHE can convert a wide variety of methyl esters to the correspondingfree acids.

The juvenile hormone esterase shows high selectivity for rapidhydrolysis of methyl and thiomethyl esters. This rapid catalyticactivity can be used to yield colorimetric assays using substrates suchas octanoic acid thiomethyl ester or methylβ-(1-pentylthio)propiothiate, methyl β-(1-pentoxy)propiothioate, andrelated β and γ homologs resulting in a catalytic marker. The highstability of juvenile hormone esterase and its rapid turnover ofthiomethyl esters also makes it an attractive reporter enzyme. Thejuvenile hormone esterase expressed in a system such as the baculovirussystem can lead to the rapid hydrolysis of a variety of methyl andthiomethyl esters. Methyl esters, thiomethylesters, or methylcarbonatescan be removed to activate drugs or pesticides such as clofibric acidmethyl ester, bifenox, butoxone methyl ester, dacthal, diclofop-methyl,chlorfenprop-methyl, or esters or carbonates of substituted2,4-dinitrophenyl uncouplers. The catalytic activity also can be used todegrade toxic materials containing a methyl ester such as pesticides inenvironmental samples or drugs or poisons such as warfarin, heroin, orbisacodyl following overdose.

We believe it likely that the biological activity comes from disruptinga fundamental process in insects that is related to turnover of nativeproteins. Thus, our mutagenesis technique is applicable to the genes ofa variety of species and to proteins extending beyond JHE.

Other Uses and Particular Uses of the JHE gene

There are advantages to using the JHE gene itself (as opposed to using acDNA for JHE) in some systems. For example, use of the gene is believedpreferable in transgenic monocots and transgenic (non-human) mammals,and the expression obtained from use of the gene itself can be higherthan with cDNA. Although insect control is the utility we presentlycontemplate as particularly preferred, the JHE gene should also beexpressed in plants, mammalian systems, bacteria, fungi, and algae, inaddition to insects and viruses.

For example, when the JHE gene is expressed in plants, then the enzymeshould act on ester-containing compounds other than insect juvenilehormone to convey herbicide resistance to the plants or may be used inbiosynthetic processes to hydrolyze methyl esters selectively. Thecatalytic activity can be used to remove methyl ester protecting groupsin specialty compound synthesis such as methyl esters of retenoic acidor chrysanthemic acid. The catalytic activity on methyl esters can beused to activate or deactivate biologically active materials with acarboxylic acid blocked by a methyl ester.

Examples of deactivation include compounds such as the sulfonyl ureaherbicides metsulfuron-methyl, tribenuron-methyl, sulfometuron-methyl,prisisulfuron, bensulfuron-methyl, or the herbicide DME2-(diphenylmethoxy)acetic acid methyl ester!. The catalytic activitycould be used to hydrolyze methyl esters critical in the biosynthesis ofnatural products. For instance, a methyl esterase will convertProtoporphyrin IX monomethyl ester to Protoporphryn IX thus reversingthe biosynthesis of the key plant pigment chlorophyll.

Expression of the JHE gene may also protect plants from invertebratesother than insects. Expression of the JHE gene by algae may be usefulfor control of mosquitos or other disease vectors.

Nucleotide sequences of this invention can be introduced into non-humanorganisms to form transgenic organisms. Introduction of the sequencesmay be double or single stranded form, and can be "sense" or"anti-sense" (that is, with the sequence reversed with respect to thesequences illustrated such as by SEQ ID NOS:1 or 2 or to the geneillustrated by FIG. 6). Shorter fragments, as small as about 16sequential bases in length, have various uses, such as in diagnosticapplications, with 16 sequential bases believed to be about the minimumlength for hybridization. The ready availability of JHE from expressionsystems of the invention, particularly from bacteria or viruses, shouldpermit other diagnostic applications, such as the use of JHE as areporter enzyme that can be coupled to antibody.

EXAMPLE 1

Animals

Larvae of Heliothis virescens were obtained from a research facility ofDow Chemical Company located at Walnut Creek, Calif.

Materials

Radioactively labeled reagents obtained from Amersham (ArlingtonHeights, Ill.). Other chemicals were of the highest quality available.Enzymes were obtained from Promega Biotec (Madison, Wis.), BoehringerMannheim Biochemicals, (Indianapolis, Ind.), United States BiochemicalCorporation (Cleveland, Ohio) and Sigma (St. Louis, Mo.).

A Bluescript plasmid with a cDNA insert from H. virescens, 3hv1(sequence not shown), 3hv21 (SEQ ID NO:1), and 3hv16 (SEQ ID NO:2), wasisolated from the E. coli host cells and host chromosomal DNA using thealkaline lysis miniprep procedure described in Maniatis et al.,Molecular Cloning, Cold Spring Harbor Laboratory (1982). The N-terminalBam HI restriction fragment from the above preparation was ligated intophage M13 (mp19). Thus, in a 25 μl volume 0.2 μg of Bam HI cut mp19 wasadded to 1.0 μg of Bluescript plasmid cut with Bam HI. After theaddition of ligase the reaction was allowed to proceed 60 minutes atroom temperature and overnight at 4° C.

Ligated DNA was used to transform competent E. coli host cells aredescribed in Rodriquez, Recombinant DNA Techniques: An Introduction,Addison-Wesley (1983). Either 0.1 μl, 1 μl or 10 μl of the ligationmixture was added to 200 μl of competent JM101. After incubation on icethe mixture was added to soft agarose at 43°-45° C. for 2 minutes. Theagarose and cells were plated with X-gal and IPTG, and incubated at 37°C. Recombinant transformants were present as white plaques.

Isolated white plaques were picked and grown for 6 hours at 37° C. in1×YT medium. Double-stranded DNA was prepared by the method described inRodriquez, supra. Briefly, the host cells were pelleted and resuspendedin a sucrose EDTA buffer. RNAse was added and the cells lysed in 1% SDSwith0.2N NaOH. The host chromosome DNA was pelleted by centrifugationand the supernatant with double-stranded RF M13 DNA removed. From thesupernatant double-stranded M13 was precipitated in isopropanol andwashed with ethanol. The double-stranded M13 was cut with Bam HI and runon a 0.8% agarose gel with Bam HI cut Bluescript plasmid to ensure thesubcloned M13 fragment was the same size as the fragment from theoriginal Bluescript plasmid.

The next step was to conduct a C-test for complementary single-strandedDNA from M13 plaques. Identification of both orientations of a clonedinsert is useful for single-strand sequencing because it is possible tosequence from both ends toward the middle. Essentially, the transformedhost cells (JM101) from independent plaques were grown for 4-6 hours in1×YT medium. Approximately 8 μl of supernatant from each plaque wasremoved and supernatant from various combinations of different plaqueswas mixed with glycerol, salt, and SDS. The mixtures were incubated at60°-70° C. for 15 minutes and allowed to cool. A sample of the mixturewas placed on a 0.8% agarose gel to detect hybridization by retardedmigration in the gel.

Single-stranded DNA from M13 with inserts in opposite orientation wasthen prepared. To do so, transformed cells were grown for 4-6 hours at37° C. Single-stranded DNA was extruded into the medium and precipitatedwith PEG and NaCl. After resuspension protein was removed with phenolchloroform. Several volumes of ethanol was added to the aqueous forprecipitation. The pellet was washed in 70% ethanol, dried andresuspended in autoclaved water.

Sequencing of the above single-stranded DNA was based on the chaintermination method of Sanger et. al, PNAS, 74, 5463 (1977). Bam HIfragments in both orientations in M13 were sequenced. For sequencingreactions, ³² P ATP was used with the reagents and instructions suppliedin the sequence kit (United States Biochemical). The sequencingreactions were run on 4% and 6% acrylamide gels. Audioradiographs of thegels were read after overnight exposure of the film to dried gel. Thecomplete Bam HI insert of approximately 840 base pairs was read. Thisincluded the putative sequence for the secretion signal peptide and theN-terminal coding sequence which corresponded to the N-terminal aminoacid sequence for JHE. The identity of the JHE cDNA insert was initiallyestablished in this manner.

EXAMPLE 2

Protein Sequencing

Juvenile hormone esterase was purified (as described by Abdel-Aal andHammock, Science, 283, pp. 1073-1076 (1986)) from the hemolymph of lastinstar larvae of Heliothis virescens. The purified preparation was seento be a single band when analyzed by electrophoresis in the presence ofSDS (Laemmli, Nature, 227, pp. 680-685 (1970)) and isoelectric focusingon a polyacrylamide gel having a ph 4.0 to pH 6.5 gradient (Pharmacia,Piscataway, N.J.). However, when subjected to Edman degradation, twoproteins were indicated to be in the preparation. The presence ofisoforms of JH esterase in H. virescens is consistent with observationsof the enzyme in other insects. From the major form was obtained areadable sequence of 35 residues. The signal from the minor formindicated a protein having a two residue extension of Ser-Ala followedby a sequence of five residues identical to the ultimate five residuesat the N-terminus of the major form. Amino acid sequencing at theN-terminal of juvenile hormone esterase was done with a Beckman 890Mliquid phase sequencer.

Probe Preparation

Both antibodies against juvenile hormone esterase and nucleotidescomplementary to the juvenile hormone esterase message were used asprobes to detect recombinant clones coding for juvenile hormoneesterase. Antisera to juvenile hormone esterase was prepared with NewZealand White female rabbits (Vaitukaitis, Methods in Enzymology(Langone and Yon Vunakis, eds.) 73, pp. 46-52, Academic Press, New York(1981)). To reduce background, the antisera was incubated overnight at4° C. with diluted Heliothis virescens hemolymph devoid of juvenilehormone esterase activity (the antisera was diluted 1:10 in a solutionof 10 mg/ml hemolymph protein in pH 7.4, I=0.2 phosphate buffercontaining 0.01% phenyl thiourea). A final dilution of 1:750 was usedfor screening. A mixture of 32 14-mer nucleotides were synthesized usinga Syntec model 1450 synthesizer. Their sequences were complementary toall the possibilities of the mRNA structure deduced from the amino acidsequence of the N-terminal. The nucleotides were purified on a Nensorb20 nucleic acid purification cartridge (Dupont Co., Wilmington, Del.)and end-labeled with ³² P with T4 polynucleotide kinase and ³² P!ATP(>6000 Ci/mmol) by a standard technique (Zeff and Geliebter, BRL Focus9-2, pp. 1-2 (1987)).

cDNA Synthesis and Cloning

Total RNA was isolated by homogenizing fat bodies in guanidiniumthiocyanate and centrifugation through cesium chloride (Turpen andGriffith, BioTechniques 4(1), pp. 11-15 (1986)). The fat bodies weredissected from last instar larvae that had been treated with epofenonane24 hours previously to increase the level of Juvenile hormone esteraseactivity (Hanzlik and Hammock, J. Biol. Chem., 262, pp. 13584-13591(1987)). At the time of treatment, the larvae weighed 200-300 mg.Polyadenylated RNA was prepared by one cycle of oligo-dT chromatography(Aviv and Leder, Proc. Nat. Acad. Sci. USA, 69, pp. 1408-1412 (1972))using oligo-dT cellulose (Collaborative Research, Cambridge, Mass.).Synthesis of cDNA from 2 μg of poly-A RNA was done by the method ofGubler and Hoffman (Gubler and Hoffman, Gene, 25, pp. 263-269 (1983))with a cDNA kit (Amersham) and a variation of its protocol. The protocolwas varied in the priming of the first strand synthesis wherein 100 ngof random hexamer primers (Pharmacia) were added 30 minutes afterinitiation of first strand synthesis by oligo-dT primers done accordingto the protocol. Size selection of the cDNA for >1350 basepairs was donewith gel permeation using a spin column (5 Prime-3 Prime, Paoli, Pa.).The size selected cDNA was treated with Eco R1 methylase, ligated to EcoR1 linkers and treated with Eco R1 restriction endonuclease according tothe protocols presented by Huynh et al. (Huynh, Young and Davis, DNACloning: A Practical Approach (Glover, D. M., ed.), pp. 49-78, IRLPress, Oxford (1984)). The free linkers were removed by three cycles ofultrafiltration using Centricon 30 microfiltrators (Amicon, Danvers,Mass.). The cDNA was then ligated to arms of a lambda gt11 expressionvector derivative (lambda-ZAP, Stratagene Cloning Systems, La Jolla,Calif.) according to the manufacturer's recommendations. The ligated DNAwas then packaged into phage heads using a two extract system (GigapackGold, Stratagene Cloning Systems), and plated on to rec E. coli hostcells (XL1-Blue, Stratagene Cloning Systems). Greater than 99% of theclones were recombinant. The cDNA library was not amplified prior toscreening.

Screening

Initial screening was done immunochemically on nitrocellulose filters towhich proteins from plated phage had been bound after induction ofprotein synthesis. Clones reacting with antibodies specific for juvenilehormone esterase were plaque-purified after detection withimmunohistochemical means (9) using a kit (Protoblot, Promega). A secondround of screening was then conducted upon plaque lifts of the isolatedclones by hybridization to the mixture of 14-mer nucleotides. This wasdone by incubating the filters with the labeled oligomers at 35° C. in5× SSPE, 3× Denhardt's solution, 100 μg/ml low molecular weight DNAafter prehybridizing in the same solution sans the oligomers. Washingwas done three times at the hybridizing temperature in a solutionconsisting of 2× SSC and 0.1% SDS. Positively reacting clones were thensubcloned into the Bluescript SK M13- plasmid (Stratagene CloningSystems) by the automatic excision process allowed by the lambda-ZAPvector.

Sequencing

Sequencing of the remaining cDNA insert, and to confirm the 840 basepair sequence, was done with the dideoxy chain termination method(Sanger, Nicklen and Coulson, Proc. Natl. Acad. Sci. USA, 74, pp.5463-5467 (1977)) using modified T7 DNA polymerase (Sequenase, UnitedStates Biochemical Corp.) and 35S labeled dATP. Templates were generatedby using both denature plasmids (Toneguzzo, Glynn, Levi, Mjolsness, andHayday, BioTechniques, 6, pp. 460-469 (1988)) and single stranded DNAfrom M13phage (Yanisch-Perron, Vieira, and Messing, Gene, 33, pp.103-119). A rec A- E. coli strain (JM109) was used for the amplificationof the subclones that were constructed with both restriction fragmentsand nested deletions (Henikoff, Gene, 28, pp. 351-359 (1984)) (kit fromStratagene Cloning Systems). Computer assisted sequence analysis wasdone with programs written by Pustell (Pustell and Kafatos, NucleicAcids Res., 12, (1984)).

Additional Library and Sequence Analysis

We decided to construct another cDNA expression library using bothrandom and oligo-dT priming of the first strand. In addition we usedcDNA selected for a size greater than 1350 bp for ligation to thevector. Screening 40,000 clones of this library produced 25immunoreactive clones, five of which hybridized to the nucleotideprobes. Three of these clones, designated 3hv1, 3hv16 and 3hv21, weresubcloned into plasmids and characterized. All three clones contained3000 bp inserts that had identical restriction patterns when incubatedwith Eco RI, Xho I and Bam HI. When the clone, 3hv21, was used as aprobe on a Northern blot, it hybridized at low stringency to a singleband with a 3.0 kb length.

The amount of screening required to isolate positive clones indicatesthat the frequency of the JH esterase message during the period of itssecretion into the hemolymph during the last instar is relatively low.

We considered the three clones (3hv1, 3hv21, and 3hv16) to be identicaldue to their identical length and restriction patterns, although slightdifferences exist as discussed hereinafter. The sequence of clone 3hv21is shown by SEQ ID NO:1 (was set out by FIG. 2 of Ser. No. 07/725,226and Ser. No. 07/265,507 of which this is a continuation-in-part andpublished in the Journal of Biological Chemistry, 264:21, pp.12419-12425 (1989). FIG. 1 shows a restriction map for this clone. Theclone was sequenced 100% in both directions. The other clone 3hv16 (SEQID NO:2) was used for baculovirus expression, mutagenesis, andbioassays.

SEQ ID NO:1 shows the cDNA sequence of JH esterase, which is a 2989 bpcDNA clone and is nearly a full length copy of the mRNA transcript.There is a short 19 base sequence prior (5') to the first ATG. Theposition and composition of the bases immediately prior to the first ATGmatches the consensus for an insect ribosome binding site except atposition -3 where a G (the second most frequent base at this site)replaces an A. After the first ATG, there is a 1714 bp open readingframe followed by an untranslated 1256 bp region including a 12 basepoly(A) tail. Translation of the open reading frame predicts a 563residue protein. The sequence and composition of the 19 residues priorto the N-terminal Trp of the secreted major form of JH esterase matchwell with the consensus for signal peptides for secretion. The molecularweight of the mature protein (sans signal peptide) is predicted to be61,012 Da, which is in agreement with the M_(r) of 62,000 derived fromelectrophoresis. The sequences of the ultimate 35 amino acids derivedfrom Edman degradation of the major form of JH esterase and thatpredicted by the cDNA sequence match except at two sites. The residuesVal 10 and Phe 33 predicted by the sequence of clone 3hv21 are indictedto be Leu and Pro, respectively, on the sequenced protein (Table 1).

                  TABLE 1                                                         ______________________________________                                        Amino acid sequence analysis of the N-terminus                                of JH esterase from H. virescens.sup.1                                        Cycle        Residue(s).sup.2                                                                        Yield.sup.3                                            ______________________________________                                         1           Trp (Ser) 1120       (230)                                        2           Gln (Ala) 780        (360)                                        3           Glu (Trp) 870        (340)                                        4           Thr (Gln) 100        (320)                                        5           Asn (Glu) 800        (350)                                        6           Ser (Thr) 560        (10)                                         7           Arg (Asn) 400        (390)                                        8           Ser       610                                                     9           Val       740                                                    10           Leu       550                                                    11           Ala       340                                                    12           His       380                                                    13           Leu       580                                                    14           Asp       590                                                    15           Ser       460                                                    16           Gly       360                                                    17           Ile       290                                                    18           Ile       390                                                    19           Arg       290                                                    20           Gly       360                                                    21           Val       300                                                    22           Pro       110                                                    23           arg       --                                                     24           Ser       130                                                    25           Ala       250                                                    26           Asp       190                                                    27           arg       --                                                     28           ile       --                                                     29           Lys       170                                                    30           phe       --                                                     31           Ala       130                                                    32           ser       --                                                     33           Pro       140                                                    34           --        --                                                     35           gly       --                                                     ______________________________________                                         .sup.1 Affinity purified JH esterase was subjected to automated Edman         degradation on a Beckman model 890M liquid phase sequencer. Derivatized       residues were confirmed with two HPLC systems employing reverse phase and     cyanocolumns. The amount of protein analyzed was 3 nmol as calculated by      Coomasie Blue dye binding.                                                    .sup.2 The initial cycles had strong secondary signals, the identity of       which are shown in parenthesis. Residues are capitalized where the            identity of the PTHamino acids were confirmed twice by elution from the       two different HPLC systems and are lower case where the identify was          assigned on the basis of elution from only one HPLC system.                   .sup.3 Yield of secondary PTHamino acids are shown in parenthesis. Hyphen     denote where the yield was not calculated for residues identified on the      basis of elution from only one HPLC system.                              

In addition, the serine present at the N-terminal of the minor form ofJH esterase protein that was sequenced is indicated to be Leu -2 on thecDNA. To answer the question of whether the differences were due to thecloning process or were genuine, we sequenced the 5' region of the twoother clones, 3hv1 and 3hv16, which were isolated from the sameunamplified library as 3hv21.

We found slight differences among all three clones. The sequence ofclone 3hv1 translates identically in the N-terminal region as clone3hv21, but differs at base 94, which is the last position of a code forserine 6. Clone 3hv16 differs at two bases (50 and 104) from clone 3hv21in the area coding for the N-terminal, one of which causes asubstitution of a phenylalanine for leucine 9 and a leucine for valine10. The substitution of isoleucine for valine at residue position 10makes the translation of clone 3hv16 match in 34 of 35 amino acidresidues determined from the purified protein.

The previous data indicated at least five slightly differenttranslations of genes for JH esterase, which suggests multiple genes oralleles for JH esterase exist in populations of H. virescens. Perhapscontributing to the heterogeneity between the cDNA's and proteinsequences is the fact that the protein and RNA were extracted from twodifferent colonies of H. virescens and represents natural variation.

There are three consensus poly(A) signal sequences (AATAAA) that startat bases 2299, 2315 and 2951. The presence of three signals forpolyadenylation may signify alternative processing of the transcript inthe 3' region. Strong evidence from studies of another noctuid mothshows that there is a constitutively expressed intracellular form of JHesterase throughout its larval stage and thus the larval stage of H.virescens. Thus a means of producing an intracellular as well as asecreted form of JH esterase is indicated to exist.

The protein translated from the cDNA clone contains four asparagineresidues, Asn 62, Asn 161, Asn 383 and Asn 496, which are candidates forglycosylation. However, preliminary evidence indicates that should thismodification be present on the secreted JH esterase from H. viresens,mannose and derivatives of glucose are not present. This informationindicates that portions of the JHE sequence can be used to make chimericesterases of altered properties.

Computer analysis of JH esterase sequence

Comparison of the translation of clone 3hv21 to protein sequences in theprotein data bank of the National Biomedical Research Foundation and totranslations of proteins characterized as esterases, lipases and serinehydrolases in GenBank revealed homologies to five proteins. Identicalresidue matches (gaps were counted as one substitution regardless oflength) of 24.2%, 23.8%, 23.2% and 23.2%, respectively, were noted tohuman pseudocholine esterase. Drosophila melanogaster acetylcholineesterase, electric ray acetylcholine esterase, and Drosophilamelanogaster esterase -6. In addition, homology to a region situatedtoward the carboxyl terminal of the large thyroid hormone precursor,bovine thyroglobulin was noted.

EXAMPLE 3

Heliothis virescens and Heliothis zea are major pest species which aredifficult to identify by visual inspection at the larval stage, yet eachspecies is most readily controlled by different insecticides. Amolecular probe for H. virescens juvenile hormone esterase was used toidentify the larval form of one species from the other.

The N-terminal 800 base pair Bam HI fragment from cloned H. virescensJHE was labelled with ³² P and hybridized to genomic DNA from H.virescens and H. zea. The DNA had been cut with the restriction enzymeXba I, the fragments separated on a 0.8% agarose gel and transferred toa nylon membrane for hybridization with the probe at 65° C.Autoradiographs of the hybridized and washed filter showed that H.virescens has at least 6 bands which are approximately 2.9, 3.4, 4.0,4.3 6.1, 7.6 and 50 kilobases. By contrast, H. zea showed none of thesebands, but has two diagnostic fragments at 2.0 and 1.5 kilobases. Thus,the JHE probe was useful as a diagnostic tool.

EXAMPLE 4

Plasmids described below are named with a"p" followed by a codeindicating their origin, and after a period the gene that they code for.Viruses are named in a similar fashion with Ac indicating that they werederived from the mNPV (multiple nuclear polyhedrosis virus) of the mothAutographa californica.

Site-directed mutagenesis

The cDNA coding sequence for JHE from clone 3hv16B (SEQ ID NO:2) wasselected for mutagenesis. This JHE clone is in the plasmid vectorpBluescriptSK+ (Stratagene) and prior to commencement of the mutagenesisreactions, the JHE coding region was removed by digestion with therestriction endonuclease Bg1II and recloned into the same vector toobtain the reverse orientation of the insert in the vector. This allowedrescue of the appropriate sense strand for site-directed mutagenesis andalso enabled the removal of the JHE coding region By both EcoRI andBg1II restriction endonucleases.

Rescue of the single strand DNA for mutagenesis was carried out bystandard techniques using the mutant M13 bacteriophage M13K07. Briefly,the JHE clone was grown in E. coli strain XL1-blue in 1 ml of LB mediumcontaining 50 μg/ml ampicillin. At early log phase, 50 μl of culture wasadded to 100 μl of M13K07 (3×108 pfu/ml) and incubated at 37° C. for 1hour then 40 μl was transferred to 20 ml of LBpmedia containing 50 μg/mlampicillin and 50 μg/ml kanamycin. The culture was incubated overnightat 37° C. and ssDNA purified from the culture supernatant as follows.The culture was centrifuged at 2,000 g for 10 minutes then thesupernatant was harvested in 16 800 μl aliquots in 1.5 ml Eppendorftubes and 200 μl of 2.5M NaCl/20% PEG added to each aliquot. After 15minutes at room temperature, the samples were centrifuged at 16,000 gfor 5 minutes and the supernatant removed. The samples were therecentrifuged for 30 seconds and residual supernatant removed. Thepellets were resuspended in 100 μl TE (10 mM Tris HCl pH 8/0.1 mM EDTA)per 4 tubes and each of the 4 aliquots was extracted once with buffersaturated phenol and twice with chloroform then precipitated withethanol. The purified ssDNA was resuspended in a final volume of 50 μlof TE.

The site-directed mutagenesis reactions were carried out by the methodof Kunkel et al. (1985) using mutant nucleotide sequences complementaryto the "rescued" coding strand obtained from the pBluescript Phagemid.We selected the sites for mutagenesis as follows. One desired mutationwas lysine (29) to arginine because this is a lysine located near theN-terminus of JHE. Another desired mutation was lysine (522) to argininebecause this is a lysine located within a potential "PEST" sequence andlocal enrichment of Pro, Ser, Glu, and Thr. A third desired mutation wasserine (201) to glycine because conserved catalytic serine motif of GlyX Ser₂₀₁ X Gly.

However, numerous options exist for modification of the JHE cDNA, orgene, to increase insecticidal activity or modify the catalyticfunctions of the enzyme. For example, it is possible to add or to removeglycosylation sites or to modify the pattern of glycosylation. For manyof the site-directed changes it is possible to add, as well as remove,regions of the enzyme that confer defined functions. Other site-directedchanges can include alteration of sites resulting in lability toproteases (intra- or extracellular), lysosome recognition sites, tissue(i.e., gut or pericardial) recognition sites, or additional sitesinvolved with ubiquitination. Sites that affect secretion or subcellulartargeting can also be modified. Endogenous modification of the enzyme,for example acylation or phosphorylation, can be a goal of mutagenesis.Larger-scale modifications of the enzyme are also possible. Forinstance, C-terminal trucated forms of JHE have already been shown toretain catalytic activity.

Generation of chimeric proteins is another possibility. One could makechimeric proteins with added peptides to provide dual catalyticactivity, increase production in an expression system, and/or changepharmacokinetic properties in a target organism. Thus it may be usefulto make a JHE/β-galactosidase enzyme or a JHE/acetyl cholinesteraseenzyme. Since X-ray analysis of esterases indicates that they exist in aC-terminal and N-terminal domain it is straightforward to make chimeraof various esterases to alter substrate specificity, kinetic properties,or pharmacokinetic properties.

The cDNA or gene may be altered to change mRNA dynamics, rate oftranscription, or rate of translation. As will be further describedhereinafter, we have also used several different promoters with the JHEcoding sequence.

Of the several separate mutations described to illustrate this aspect ofthe invention, one we designated K29R (lysine 29 mutated to arginine),one as K522R (lysine 522 mutated to arginine), and one as S201G (serine201 mutated to glycine). All mutations were confirmed by double-strandedsequencing using a sequenase kit (USB).

The K29R, K522R, and S201G JHE mutants were transferred directly to thebaculovirus transfer vector pAcUW21 (FIG. 2) by digestion with Bg1II,gel purification of the JHE coding region, and cloning into the Bg1IIsite of the baculovirus transfer vector pAcUW21. A double mutant of JHE(K29R, K522R) was constructed by removal of the N-terminal half of JHE(containing lysine 29) from the pAcUW21.JHE-K522R construct andreplacing this with the N-terminal half of JHE from the pAcUW21.JHE-K29Rconstruct. This was done by digesting pAcUW21.JHE-K522R with BamHI whichcuts the clone at position 851 in JHE and also cuts the pAcUW21 vectorwithin the polyhedrin gene. This region was then replaced with theequivalent region from pAcUW21.JHE-K29R to regenerate the same vectorand clone but containing the K29R mutation. The correct orientation ofthe modified JHE sequence was confirmed in each case by restrictionanalysis. Transformation, plasmid preparation and digestions werecarried out using standard techniques (Maniatis et al., 1990). Thepresence of the K29R and K522R mutations were confirmed by digestionwith restriction endonucleases. The presence of the S201G mutation wasconfirmed by sequencing.

Virus Constructs

The viruses AcUW21.JHE-K29R, AcUW21.JHE.K522R, AcUW21.JHE-K29R,K522R andAcUW21.JHE-S201G (henceforth referred to as AcJHE-29, AcJHE-522,AcJHE-29,522 and AcJHE-201 respectively) were made by cotransfection ofcell line IPLB Sf-21 of Spodoptera frugiperda with polyhedron negativeAutographa californica nuclear polyhedrosis virus (AcNPV) DNA with therespective plasmids with DNA purified from the polyhedrin negative virusAcRP8 (Matsuura et al., 1987) using Lipofectin Gibco. Homologousrecombination between the recombinant plasmids and the vital DNAresulted in polyhedrin positive recombinant viruses against a backgroundof polyhedrin negative non-recombinant virus. Additional cell lines andtypes of media can be used to alter the production of JHE in vitro.

Virus Purification

Recombinant polyhedrin positive viruses were purified to homogeneity bysequential plaque assays using standard assay procedures. The expressionof JHE by recombinant viruses was confirmed by JHE activity and SDS-PAGEfor the JHE-29,522 mutant and by SDS-PAGE and Western blotting for theJHE-201 mutant.

Propagation of viruses was carried out in Spodoptera frugiperda cells(Sf) IPLB Sf-21 (Vaughn et al., 1977) in ExCell 400 medium (JRHBioSciences) containing 1% Penicillin-Streptomycin (typically containing100 μg/ml penicillin G and 100 μg/ml streptomycin), at 28° C. Plaqueassays were carried out as described by Brown and Faulkner (1977).Purified virus was amplified by larval infection of Heliothis virescens.Virus was purified from cadavers by homogenization in double deionizedwater and differential centrifugation at 4° C. Virus was stored at 4° C.in 0.02% sodium azide. Azide was removed by washing before use of virusin bioassays.

The recombinant virus AcUW2(B).JHE (henceforth referred to as AcJHE),engineered to express the unmodified JHE, and the wild typenon-engineered virus AcNPV C6 were also used for comparative purposes.

Baculovirus expression of JHE in vitro

Petri dishes of Sf-21 cells were set up at 1.5×106 cells/ml at 28° C.,and infected 24 hours later with AcJHE, AcJHE-29, AcJHE-522 orAcJHE-29,522 at 10 pfu/cell. Expression levels of the modified JHEsattained in vitro in Sf cells were monitored at 6 to 12 hour intervalsby colorimetric assay for JHE activity (McCutchen, unpublished), foreach modified JHE.

Kinetic parameters (Km and Vmax) were determined for JHE-29,522 usingtritiated JH III, or C₆ H₁₃ OCH₂ C(O)SCH₃ as substrate, to ensure thatthese were not altered by the modifications of the JHE coding sequence.Kinetic parameters had been determined previously for JHE, JHE-29 andJHE-522. Crude medium containing baculovirus expressed enzyme was usedfor these studies.

Bioassay

Second instar larvae of Trichoplusia ni or H. virescens were infected atvarious doses of test or control virus to determine the lethal dose.Diet plugs of uniform size (made with a Pasteur pipette) were inoculatedwith polyhedra (five doses of between 120 to 7 polyhedrin inclusionbodies). Mid-second instar larvae were allowed to feed on the treateddiet plugs in 96 well microtitre plates for 24 hours, 50 larvae beinginfected per dose of virus. After 24 hours, those that had completelyconsumed the diet plug and therefore ingested the required dose, weretransferred to individual tubs of diet and maintained at 24° C. for T.ni and 27° C. for H. virescens. Controls were mock infected. AcNPV C6was used as a reference virus for comparison with previous bioassay datafor other engineered viruses where the same reference virus was used forreference (McCutchen et al., 1991; Stewart et al., 1991; Merryweather etal., 1990). Mortality was scored after 9 days. Bioassays were replicated3 times and lethal doses calculated using the POLO computer program(Russell et al., 1977).

Neonate T. ni or H. virescens were infected using the droplet feedingassay (Hughes et al., 1986) at 2×10⁶ pibs/ml to determine the lethaltime for each virus. Fifty larvae were infected with each virus.Mortality of the larvae was scored every 4,6 or 8 hours according to themortality rate. LT₅₀ values were determined using the POLO probitanalysis program. Lethal Ratios for Time (LRT) were determined usingAcNPV C6 as the reference strain, for comparison with other datagenerated in different laboratories using different bioassay techniquesand conditions.

Second instar larvae of Manduca sexta were injected with 2×10⁴ pfu (2μl) of each recombinant virus (AcJHE, AcJHE-29, AcJHE-522 or AcJHE-29,522)and bled at 24 hour intervals post infection to assess the level ofmodified JHE expression in vivo. Five larvae were bled per time pointper treatment. Wild type virus AcNPV C6) was injected as control. Mockinfected controls injected with media (ExCell 401) were also analyzed.Hemolymph samples were diluted in PBS containing 100 μg/ml BSA andstored at -20° C. prior to assay for JHE activity (Hammock and Roe,1985).

Pharmacokinetics

In order to establish whether the modifications made to the JHE affectedthe rate of uptake of the enzyme from the hemolymph by the pericardialcells, pharmacokinetic analysis was carried out. Wild type and modifiedJHEs harvested from cell culture were purified by DEAE anion exchange.JHE (1 nm/min) was injected into second instar M. sexta (30 to 38 mg)and 6 larvae per time point per treatment bled 0.3, 1, 2, 3, and 4 hourspost injection. JHE activity in hemolymph samples was determined byradiochemical assay. Half lives were determined by exponentialregression analysis, and rate constants of clearance calculated.

Construction of recombinant viruses expressing modified JHE

The specific alterations made in the modified JHE sequences wereconfirmed by sequencing. The coding sequences were introduced into thebaculovirus transfer vector, pAcUW21 (FIG. 2), and the correct insertionconfirmed by restriction analysis. Sf-21 cells were cotransfected withthe appropriate transfer vector and AcRP8 DNA. The recombinant viruseswere purified by 4 rounds of plaque purification by screening forpolyhedrin positive plaques and JHE activity.

Baculovirus expression in vitro

All three mutated viruses expressing modified JHE (AcJHE-29, AcJHE-522and AcJHE-29,522) produced active enzyme in cell culture (FIG. 3),although expression of JHE-29,522 was lower than expression of JHE,JHE-29 or JHE-522.

We found that JHE mutants having alterations at sites presumed to beinvolved with proteolytic degradation by the ubiquitination pathway ledto greatly improved insecticidal efficiency. That is, site-directedmutagenesis significantly improved the speed of kill by baculovirusescontaining some of the modified JHEs in both H. virescens and T. ni. Inparticular, we found larvae injected at second instar with AcJHE-29,AcJHE-522 or AcJHE-29,522 gave high level expression of JHE from 96hours post infection (FIG. 5). Only low levels of JHE activity (lessthan 1 nm/min/ml) were detected in hemolymph from uninfected controllarvae (injected with medium) and larvae infected with wild type virusor AcJHE.

Lethal doses were determined using the POLO program. For both H.virescens and T. ni, the LD50 values are comparable to those of the wildtype virus AcNPV C6. The LD50 for AcJHE-29,522 is similar to that of thecontrol virus AcJHE.

LT₅₀ data (Table 2) show that the virus AcJHE-29,522 has significantlyimproved LT₅₀ of 84 hours in T. ni and 80 hours in H. virescens,compared to 106 and 114 hours for AcNPV C6 respectively. AcJHE-29 andAcJHE-522 give slower kill than AcNPV C6 in T. ni, and quicker kill inH. virescens.

                  TABLE 2                                                         ______________________________________                                        Biological Activity (LT.sub.50 -hours) of                                     Mutant JHE Expressed in AcNPV                                                                T. ni                                                                              H. virescens                                              ______________________________________                                        AcNPV C6         106    114                                                   AcJHE            107    118                                                   AcJHE-29         117    106                                                   AcJHE-522        120    107                                                   AcJHE-29, 522     84     80                                                   AcJHE-201         88     82                                                   ______________________________________                                    

EXAMPLE 5

The basic protein is a 6.9K, arginine-rich polypeptide, expressed duringthe late phase of viral infection (10-20 hours post infection). We usedthe basic protein promoter in place of the polyhedrin gene in theplasmid transfer vector pAcMP1 by following a procedure described byHill-Perkins and Possee, Journal of General Virology, 71, pp. 971-976(1991). We ligated the coding sequence for juvenile hormone esterasefrom Heliothis virescens into the pAcMP1 transfer vector at the Bg1 IIcloning site, to produce a plasmid we called "pAcMP1JHE."

Cotransfection of the plasmid pAcMP1JHE with DNA from the virus AcUW1-PH(by the general procedure described by Weyer et al., Journal of GeneralVirology, 71, pp. 1525-1534 (1990)) resulted in a basicprotein-positive, polyhedrin-positive, p10-negative virus, thepolyhedrin being under control of the p10 promoter at the p10 locus. JHEis expressed under control of the duplicated basic protein promoter inthis virus (AcMP1JHE).

The p10 protein is expressed at high levels during the very late phaseof virus infection. The p10 promoter has been inserted at the polyhedrinlocus in the transfer vector pAcUW2(B) (Weyer et al., J. Gen. Virol.,71, pp. 1525-1534 (1990)). We inserted the sequence for juvenile hormoneesterase at the Bg1 II cloning site to produce the plasmidpAcUW2(B).JHE. Cotransfection of this plasmid with DNA from AcRP8produced a polyhedrin-positive, p10-positive virus, AcUW2(b)JHE,expressing JHE under control of a duplicated p10 promoter.

Turning to FIG. 4, one can see that the just-described plasmid pAcMP1JHE(that is, with the basic protein promoter) gave enhanced expressionlevels of JHE with respect to levels from the polyhedrin promoter andeven with respect to levels achieved with the p10 promoter.

EXAMPLE 6

Library construction and screening

DNA was prepared from embryonic tissue derived from an outbredlaboratory population of Heliothis virescens. In order to produce highmolecular weight DNA care was taken to avoid shearing (Kaiser and Murray1985), and consequently, the size of the purified DNA was larger thanbacteriophage T4 DNA (>170 kb). The genomic library was constructed asdescribed by Maniatis et al. (1982) and Kaiser and Murray (1985).Specifically, an optimized MboI digestion was used to partially digestthe genomic DNA. The digested DNA was separated on a sucrose gradientand size-selected by pooling fractions corresponding to 10-20 kbfragments. Lambda EMBL3 arms (Stratagen) and the size-selected genomicDNA were ligated and then packaged with a commercial extract (GigapakGold, Stratagene).

Escherischia coli strain LE392 (rec A⁺) was used to plate the library ata density of approximately 30,000 pfu per 150×15 mm petri plate. Plaqueswere lifted in duplicate onto nitrocellulose disks, which were preparedfor hybridization by standard protocols. To screen the library, a 850 bpN-terminal Bam HI fragment from the JHE cDNA (Hanzlik et al., 1989) waslabelled by random priming using hexamers (pdN6, Pharmacia) and ³² P(Amersham). After hybridization, the last wash was in 0.1× SSC at 55° C.The washed and dried filters were overlaid with XAR film (Kodak) and anintensifying screen for overnight exposure at -70° C. Positive clones,detected as duplicate signals on developed film, were isolated by astandard plaque purification protocol. A phage stock was prepared foreach clone and used in the process of purifying lambda DNA on cesiumchloride gradients (Maniatis et al., 1982).

Clone characterization

A hybridization experiment was conducted to determine how much of theJHE cDNA sequence was present in each of the JHE positive genomicclones. Probes from the JHE cDNA were hybridized to 5 clones which hadbeen dot-blotted onto nylon membranes. Hybridization was performed in 7%sodium dodecyl sulfate, 1 mm EDTA and 0.5M NaPO₄ at 60° C. Afterhybridization the last wash was in 0.1× SSC at 65° C.

For further analysis, an 11 kb KpnI fragment (C11K) from genomic clone Cwas subcloned into pUC118. To confirm that clone C11K contained all ofthe JHE gene, and to aid restriction mapping of the gene, syntheticnucleotide primers were made to the sequence of the 5' and 3' ends ofthe full length JHE cDNA clone previously described in the parentapplications and their former FIG. 2. (By "former" FIG. 2, we mean FIG.2 of Ser. No. 07/725,226 and its predecessor Ser. No. 5 07/265,507, ofwhich this application is a continuation-in-part. The former FIG. 2 wasfound to contain several minor errors in sequence, which have beencorrected in the present SEQ ID NO:1.) Primer b corresponds to bases29-46 of the JHE cDNA sequence and primer f corresponds to bases2775-2794. A sequence kit (United States Biochemical) was used for dsDNAsequencing.

A map of clone C11K was generated to verify the linear correspondence ofrestriction sites, in relationship to the JHE cDNA, and for detection ofintrons within the genomic clone. From the 5' sequencing of C11K an NaeIrestriction site was located upstream of the reading region and used torestriction map the 5' end of the gene. The 3' sequence of the genomicclone confirmed the presence of an XhoI site which was useful formapping the 3' end of the gene. Using NaeI and XhoI sites as referencepoints, a map was constructed by multiple restriction endonucleasedigestions with Bg1 II, ClaI, NaeI, NcoI, KpnI, PstI, and XhoI. From therestriction analysis it was possible to approximate the position ofintrons, which were defined by synthesizing nucleotide primerscomplementary to the cDNA and sequencing across the intron/exonjunctions.

Library construction and screening

Hybridization of the labelled JHE cDNA BamHI fragment yielded 6 positiveclones in a screen of approximately 250,000 primary plaques from thegenomic library. Only one of the genomic clones, C, hybridized to eachof the cDNA probes and was assumed to contain most, if not all, of theJHE gene. C11K, the 11 kb Kpn fragment, was found to contain the entireJHE gene by sequence homology to the 5' and 3' termini of the JHE cDNA

FIG. 6 summarizes the JHE gene, which is approximately 11 kb with fourintrons. Restriction sites used for the mapping of subclone C11K arerepresented. The exons are represented by solid regions and the intronsare represented by stippling. Regions upstream and downstream of thecoding region are shown as empty rectangles. The exact location ofintron/exon boundaries were determined by sequencing the gene in theregions shown by restriction mapping to have introns. Primerscomplimentary to JHE cDNA allowed sequencing of most of the intron/exonjunctions. To facilitate the sequencing of the junctions at positions 53and 519, C11K was subcloned into two fragments at the NcoI site. Forwardand reverse vector sequencing primers were then used to sequence out tointron junctions on either side of the NcoI site (*). The wedge next tothe letter designation of each primer indicates the sequencingorientation from the primer. There is an ordered correspondence betweenrestriction sites in clone C11K and sites in the JHE cDNA, indicatingthat the clone is representative. Four introns occur in the codingregion of the gene and none are found in the 3' untranslated region. A4.3 kb intron is located in the 19 amino acid leader sequence region,between cDNA bases 52 and 53. The possible significance of a largeintron located close to the 5' end of the gene is not clear, but ananalogous situation is found in the Bombyx mori fibroin gene. The otherthree introns are relatively small, ranging in size from 154 bp to 204bp. The consensus sequences (GT-AG), common to the beginning and end ofalmost all introns, are present in the JHE gene.

The foregoing examples illustrate certain embodiments of the presentinvention, and are not intended to limit the scope of the invention,which is defined in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2989 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCCCACCGAACAGACATGACTTCACACGTACTCGCGCTCGCCTTCCTTCTACACGC60                GTGCACAGCGCTGGCGTGGCAGGAGACAAATTCGCGCAGCGTGGTCGCCCATCTGGACTC120               CGGCATTATACGCGGCGTGCCGCGCTCAGCGGATGGCATCAAGTTCGCCAGCTTCCTAGG180               AGTGCCCTACGCTAAGCAGCCTGTTGGAGAACTCAGGTTTAAGGAGCTCGAGCCTCTAGA240               ACCTTGGGATAATATCCTGAACGCAACAAATGAAGGACCCATCTGCTTCCAAACAGATGT300               ATTATACGGGAGGCTCATGGCGGCAAGCGAGATGAGCGAGGCTTGCATATACGCCAACAT360               TCATGTTCCATGGCAAAGCCTTCCCCGAGTGAGGGGGACCACACCTTTACGGCCTATCCT420               GGTGTTCATACATGGTGGAGGATTTGCTTTCGGCTCCGGCCACGAGGACCTACACGGACC480               AGAATATTTGGTCACTAAGAATGTCATCGTCATCACGTTTAATTACAGATTGAACGTCTT540               CGGTTTCCTGTCCATGAACACAACAAAAATCCCCGGGAATGCCGGTCTCCGGGATCAGGT600               AACCCTGTTGCGCTGGGTGCAAAGGAACGCCAAGAATTTCGGAGGAGACCCCAGCGACAT660               CACCATAGCGGGGCAGAGCGCTGGTGCATCAGCTGCGCATCTACTGACTCTTTCTAAAGC720               TACTGAAGGTCTTTTCAAAAGAGCGATTCTGATGAGCGGAACAGGAATGAGCTACTTCTT780               TACTACTTCTCCACTTTTCGCGGCCTACATTTCGAAACAGTTGTTGCAAATCCTGGGCAT840               CAACGAGACGGATCCCGAAGAAATACATCGGCAGCTCATCGACCTACCCGCAGAGAAACT900               GAACGAGGCTAACGCCGTCCTGATTGAACAAATTGGCCTGACAACCTTCCTCCCTATTGT960               GGAATCCCCACTACCTGGAGTAACAACCATTATTGACGATGATCCAGAAATCTTAATAGC1020              CGAAGGACGCGGCAAGAATGTTCCACTTTTAATAGGATTTACCAGCTCAGAATGCGAGAC1080              TTTCCGCAATCGACTATTGAACTTTGATCTCGTCAAAAAGATTCAGGACAATCCTACGAT1140              CATAATACCGCCTAAACTGTTATTTATGACTCCACCAGAGCTGTTGATGGAATTAGCAAA1200              GACTATCGAGAGAAAGTACTACAACGGTACAATAAGTATCGATAACTTCGTAAAATCATG1260              TTCAGATGGCTTCTATGAATACCCTGCATTGAAACTGGCGCAAAAACGTGCCGAAACTGG1320              TGGAGCTCCACTGTACTTGTACCGGTTCGCGTACGAGGGTCAGAACAGCATCATCAAGAA1380              GGTAATGGGGCTGAACCACGAGGGTGTCGGCCACATTGAGGACTTAACCTATGTGTTTAA1440              GGTCAACTCTATGTCCGAAGCTCTGCACGCATCGCCTTCTGAGAATGATGTGAAAATGAA1500              GAATCTAATGACGGGCTATTTCTTAAATTTTATAAAGTGCAGTCAACCGACATGCGAAGA1560              CAATAACTCATTGGAGGTGTGGCCGGCTAACAACGGCATGCAATACGAGGACATTGTGTC1620              TCCCACCATCATCAGATCCAAGGAGTTCGCCTCCAGACAACAAGACATTATCGAGTTCTT1680              CGACAGCTTCACCAGTAGAAGCCCGCTTGAATGATAAGACTGAACTATTGTCATCGATAT1740              AAATATGTTGTTAATGTTAGTTAAGAGTTCTCATAGTGCAGTGAGCGTTTGAACTGAACC1800              ACTGGTCTCAGAAGATCGAAGTTTCATCCTATGACATAAGAGTGTACAATGTTTTCAGTT1860              AAGTGTTGATGTTGATACTTTAATTTGCATTAATTTATTTAGAGTAAGGTTAATGTCACA1920              AGTCTAGTCGGTTACTTAAGTAATTTCTTGCCAACATTGGTGTAATGCCTTTTCGTTGAG1980              TTTCAAAAAATATTAATATTATATGCATTATAAATTAAATTCTAATTTTCATCGTAGAAT2040              ATAATACCATAGTTAGCATTGTTGCTCTTTGAGAAGAGGTCAATGCCCAGCAATAGGAAA2100              GTACAAAGGTCGATGATGATGAATAAGCAGATAAATTATAGAGCTTCTACTTCATTGATG2160              TTGATTGAAACTCATGTTGACATCTTTGTGAAATCATTTGACATCAAAGAGAACATAACT2220              TTAGTTTAACGACACGGATTTACTATTAGGAACAGCTAGACCTTCTTTAGACCTAGTATT2280              GTTTTACGAAGCAATTGTAATAAAACTTGGGTGAAAATAAAGGTTAGTCGTAATTACAGC2340              ATTACGACTAAGCTTTGTTAGTGCCCGGAAGATTGATCTCATAAAACTACACTAGGCTAT2400              GGATAACAATCCGCCCGCAATTTAATTTTAAGTTAATATAAGTTATTTTGAAAATTATAT2460              TTTTGTACAAAATGCTGCAGATCACGGGACGTCTATTCGATTTGATATTCGAAAAGGAAT2520              TTTACTATTTTGACTTTCGAGAGTCTGACGAGATGTTAGTATATTCGCGAGCATCCATAA2580              ATCGAATTTGTGTTAATTGGAAGTTCGTTCTCGATCTAGATTCGTAAGGTGCATGGTGCT2640              ACTTACTAGATAAATATTAGCAATACAATTGAATTTCGTATTCCAAAACTATCCCTATTC2700              CTGATTACGAAGGGCAGTGTACAAAATAGTGAAAAATTGTAATTGTACAGAATGATAATC2760              CCGTGATCCAAGCACTCGAGATGCGTAATGAAGCGACTGATGTAACGTATTATAATTTAA2820              GTCAATTTACTATTAGTTTTCAACGCCTTTGTAAATATTTCACTTTCTAATGTAATTTTA2880              GTATTCCCGCACAATGACGCCAGAGTACAATGATCGGACGCGATCGCGTGGCGTTACATT2940              TAATGATTCAAATAAATAATTGCGTCGGACGGACGTGAAAAAAAAAAAA2989                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3047 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAATTCCAACAGACATGACTTCACACGTACTCGCGCTCGCCTTCTTTCTACACGCGTGCA60                CAGCGCTGGCGTGGCAGGAGACAAATTCGCGCAGCGTGCTCGCCCATCTGGACTCCGGCA120               TTATACGCGGCGTGCCGCGCTCAGCGGATGGCATCAAGTTCGCCAGCTTCCTAGGAGTGC180               CCTACGCTAAGCAGCCTGTTGGAGAACTCAGGTTTAAGGAGCTCGAGCCTCTAGAACCTT240               GGGATAATATCCTGAACGCAACAAATGAAGGACCCATCTGCTTCCAAACAGATGTATTAT300               ACGGGAGGCTCATGGCGGCAAGCGAGATGAGCGAGGCTTGCATATACGCCAACATTCATG360               TTCCATGGCAAAGCCTTCCCCGAGTGAGGGGGACCACACCTTTACGGCCTATCCTGGTGT420               TCATACATGGTGGAGGATTTGCGTTCGGCTCCGGCCACGAGGACCTACACGGACCAGAAT480               ATTTGGTCACTAAGAATGTCATCGTCATCACGTTTAATTACAGATTGAACGTCTTCGGTT540               TCCTGTCCATGAACACAACAAAAATCCCCGGGAATGCCGGTCTCCGGGATCAGGTAACCC600               TGTTGCGCTGGGTGCAAAGGAACGCCAAGAATTTCGGAGGAGACCCCAGCGACATCACCA660               TAGCGGGGCAGAGCGCTGGTGCATCAGCTGCGCATCTACTGACTCTTTCTAAAGCTACTG720               AAGGTCTTTTCAAAAGAGCGATTCTGATGAGCGGAACAGGAATGAGCTACTTCTTTACTA780               CTTTCTCCACTTTTCGCGGCCTACATTTCGAAACAGTTGTTGCAAATCCTGGGCATCAAC840               GAGACGGATCCCCGAAGAAATACATCGGCAGCTCATCGACCTACCCGCCGAGAAACTGAA900               CGAGGCTAACGCCGTCCTGATTGAACAAATTGGCCTGACAACCTTCGTCCCTATTGTGGA960               ATCCCCACTACCTGAAGTAACAACCATTATTGACGATGATCCAGAAATCTTAATAGCCGA1020              AGGACGCGGCAAGAATATTCCACTTTTAATAGGATTTACCAGCTCAGAATGCGAGACTTT1080              CCGCAATCGACTATTGAACTTTGATCTCGTCAAAAAGATTCAGGACAATCCTACGATCAT1140              AATACCGCCTAAACTGTTATTTATGACTCCACCAGAGCTGTTGATGGAATTAGCAAAGAC1200              TATCGAGAGAAAGTACTACAACGGTACAATAAGTATCGATAACTTCGTAAAATCATGTTC1260              AGATGGCTTCTATGAATACCCTGCATTGAAACTGGCGCAAAAACGTGCCGAAACTGGTGG1320              AGCTCCACTGTACTTGTACCGGTTCGCGTACGAGGGTCAGAACAGCATCATCAAGAAGGT1380              AATGGGGCTGAACCACGAGGGTGCCGGCCACATTGAGGACTTAACCTACGTGTTTAAGGT1440              CAACTCTATGTCCGAAGTTCTGCACGCATCGCCTTCTGAGAATGATGTGAAAATGAAGAA1500              TCTAATGACGGGCTATTTCTTAAATTTTATAAAGTGCAGTCAACCGACATGCGAAGACAA1560              TAACTCACTGGAGGTGTGGCCGGCTAACAACGGCATGCAATACGAGGACATTGTGTCTCC1620              CACCATCATCAGATCCAAGGAGTTCGCCTCCAGACAACAAGACATTATCGAGTTCTTCGA1680              CAGCTTGTCCAGTAGAAGCCCACTTGAATGATAAGACTGAACTATTGTCATCGATATAAA1740              TATGTTGTTAATGTTAGTTAAGAGTTCTCATAGTGCAGTGAGCGTTTGAACTGAACCACT1800              GGTCTCAGAAGATCGAAGTTTCATCCTATGACATAAGAGTGTACAATGTTTTCAGTTAAG1860              TGTTGATGTTGATACTTTAATTTGCATTAATTTATTTAGAGTAAGGTTAATGTCACAAGT1920              CTAGTCGGTTACTAAAGTAATTTCTTGCCAACATTGGTGTAATGCCTTTTCGTTGAGTTT1980              CAAAAAATATTATTATTATATGCATTTTAAATTAAATTCTAATTTTCATCGTAGAATACA2040              ATACCATAGTTAGCATTGTTGCTCTTTGAGAAGAGGCCAATGCCCAGCAATAGGAAAGTA2100              CAAAGGTCGATGATGATGAATAAGCAGATAAATTATAGAGCTTCTACTTCATTGATATTG2160              ATTGAAACTCATGTTGACATCTTTGTGAAATCATTTGACATCAAAGAGAACATAACTTTA2220              GTTTAACGACACGGATTTACTATTGAAACAGCTAGACCTTCTTTAGACCTAGTATTGTTT2280              TACGAAGCAATTGTAATAAAACTTGGGTGAAAATAAAGGTTAGTCGTAATTACAGCATTA2340              CGACTAAGCTTTGTTAGTGCCCGGAAGATTGATCTCATAAAACTACACTAGGCTATGGAT2400              AACAATCCGCCCGCAATTTAATTTTAAGTTAATATAAGTTATTTTGAAAATTATATTTTT2460              GTACAAAATGCTGCAGATCACGGGACGTCTATTCGATTTGATATTCGAAAAGGAATTTAA2520              CTATTTTGACTTTCGAGAGTCTGACGTGATGTTAGTATATTCGCGAGCATCCATAATTAA2580              CTATTTTGACTTTCGAGAGTCTGACGTGATGTTAGTATATTCGCGAGCATCCATAAATCG2640              AATTTGTGTTAATTGGAAGTTCGTTCTCGATCTAGATTCGTAAGGTGCATGGTGCTACTT2700              ACTAGATAAATATTAGCAATACAATTGAATTTCGTATTCCAAACGAAGGGCAGTGTACAA2760              AATAGTGAAAAATTGTAATTGTACAGAATGATAATCCCGTGATCCAAGCACTCGAGATGC2820              GTAATGAAGCGACTGATGTAACGTATTATAATTTAAGTCAATTTACTATTAGTTTTCAAC2880              GCCTTTGTAAATATTTCACTTTCTAATGTAATTTTAGTATTCCCGCACAATGACGCCGAG2940              TACAATGATCGGACGCGATCGCGTGGCGTTACATTTAATGATTCAAATAAATAATTGCGT3000              CGGACGGACGTGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3047                           __________________________________________________________________________

It is claimed:
 1. A recombinant virus having a nucleotide sequence for ajuvenile hormone esterase or a mutant thereof, wherein the expression ofthe nucleotide sequence results in juvenile hormone esterase or a mutantthereof capable of degrading insect juvenile hormone with a k_(cat)/K_(m) ratio greater than about 10⁸, the nucleotide sequence being as inSEQ ID NO:1 or SEQ ID NO:2 or a mutated sequence thereof, the mutatedsequence coding for a substituted amino acid reside at one or more oflysine positions 29 and 522 or serine
 201. 2. The recombinant virus asin claim 1 wherein the virus is a baculovirus.
 3. The recombinant virusas in claim 2 wherein the baculovirus is a nuclear polyhedrosis virus.4. The recombinant virus as in claim 2 wherein the baculovirus isAutographa californica.